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抗钠- 钙交换体-1 重复序列(117-137)抗体对心肌钠- 钙交换电流及钙电流的抑制作用

封启龙, 武冬梅, 崔香丽, 赵画晨, 林媛媛, 赵录英, 吴博威

山西医科大学1 生理学系； 药理教研室，太原 030001； 细胞生理学省部共建教育部重点实验室，太原 030001； 北京脐带血库，北京 100176

摘要

本研究旨在探讨抗钠-钙交换体(Na+-Ca2+ exchanger, NCX) α-1 重复序列(117-137)抗体对大鼠心肌细胞钠- 钙交换电流的影响。实验用合成的α-1 重复序列(117-137)肽免疫大鼠制备抗α-1 重复序列(117-137)抗体；应用全细胞膜片钳技术，在急性分离的心肌细胞上观察抗α-1 重复序列(117-137)抗体对大鼠心肌细胞钠- 钙交换电流的影响，同时还观察了其对大鼠心肌细胞L 型钙通道、电压门控钠通道和豚鼠心肌延迟整流钾通道电流的影响；最后，采用EMBOSS Pairwise Alignment Algorithms软件对NCX α-1 重复序列和L 型钙通道(1 076~1 096)的氨基酸序列进行了比较。结果显示，抗α-1 重复序列(117-137)抗体对大鼠心肌细胞Na+-Ca2+ 交换电流呈现剂量依赖性的抑制作用。在钳制电压为+50 和100 mV 时，其抑制外向和内向钠- 钙交换电流的IC50 分别为18.9 和22.4 nmol/L。此外，该抗体还对L 型钙通道电流具有抑制效应(IC50=22.7 nmol/L)，对电压门控钠通道和延迟整流钾通道电流则无明显影响。通过氨基酸序列比对发现，NCX α-1 重复序列(117-137)与L 型钙通道IIIS5-S6序列(孔环)之间具有23.8% 的相似性。以上结果表明，抗NCX α-1 重复序列(117-137)抗体是心肌NCX 的一种抑制性抗体，同时也可以抑制L 型钙通道。

关键词： 钠- 钙交换体; 膜片钳技术; 心肌细胞; 抗体

分类号：Q46

Inhibitory effects of purified antibody against -1 repeat (117-137) on Na+-Ca2+ exchange and L-type Ca2+ currents in rat cardiomyocytes

FENG Qi-Long, WU Dong-Mei, CUI Xiang-Li, ZHAO Hua-Chen, LIN Yuan-Yuan, ZHAO Lu-Ying, WU Bo-Wei

Department of Physiology； Department of Pharmacology, Shanxi Medical University, Taiyuan 030001, China； 3Key Laboratory of Cellular Physiology, Shanxi Province and Ministry of Education, Taiyuan 030001, China； 4Beijing Cord Blood Bank, Beijing 100176, China

Abstract

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na+-Ca2+ exchanger (NCX), it ispossible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is toinvestigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against thesynthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na+-Ca2+ exchange current (INa/Ca) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-typeCa2+ current (ICa,L), voltage-gated Na+ current (INa) and delayed rectifier K+ current (IK) were also observed. The amino acid sequences ofα-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca2+ channel were compared using EMBOSS Pairwise AlignmentAlgorithms. The results showed that outward and inward INa/Ca were decreased by the antibody against α-1(117-137) dose-dependentlyin the concentration range from 10 to 160 nmol/L, with IC50 values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, theantibody also decreased ICa,L in a concentration-dependent manner with IC50 of 22.7 nmol/L. No obvious effects of the antibody on INaand IK were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCXα-1(117-137) and residues 1 076-1 096 within L-type Ca2+ channel. These results suggest that antibody against α-1(117-137) is a blockingantibody to NCX and can also decrease ICa,L in a concentration-dependent manner, while it does not have obvious effects on INa and IK.

Key words: sodium-calcium exchanger; patch-clamp technique; cardiac myocytes; antibody

收稿日期：2010-06-24　　录用日期：2010-09-14

通讯作者：吴博威　　E-mail: wubowei299@sohu.com

引用本文：

封启龙, 武冬梅, 崔香丽, 赵画晨, 林媛媛, 赵录英, 吴博威. 抗钠- 钙交换体-1 重复序列(117-137)抗体对心肌钠- 钙交换电流及钙电流的抑制作用[J]. 生理学报 2010; 62 (5): 407-414.

FENG Qi-Long, WU Dong-Mei, CUI Xiang-Li, ZHAO Hua-Chen, LIN Yuan-Yuan, ZHAO Lu-Ying, WU Bo-Wei. Inhibitory effects of purified antibody against -1 repeat (117-137) on Na+-Ca2+ exchange and L-type Ca2+ currents in rat cardiomyocytes. Acta Physiol Sin 2010; 62 (5): 407-414 (in Chinese with English abstract).